The invention relates to new antifibrillogenic agents, a composition containing same and a method of using these new antifibrillogenic agents for inhibiting amyloid fibril formation.
Amyloidosis refers to a pathological condition characterized by the presence of amyloid fibers. Amyloid is a generic term referring to a group of diverse but specific protein deposits (intracellular and/or extracellular), which are seen in a number of different diseases. Though diverse in their occurrence, all amyloid deposits have common morphologic properties, stain with specific dyes (e.g., Congo red), and have a characteristic red-green birefringent appearance in polarized light after staining. They also share common ultrastructural features and common x-ray diffraction and infrared spectra.
Amyloid-related diseases can either be restricted to one organ or spread to several organs. The first instance is referred to as “localized amyloidosis” while the second is referred to as “systemic amyloidosis”.
Some amyloidotic diseases can be idiopathic, but most of these diseases appear as a complication of a previously existing disorder. For example, primary amyloidosis can appear without any other pathology or can follow plasma cell dyscrasia or multiple myeloma. Secondary amyloidosis is usually seen associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis). A familial form of secondary amyloidosis is also seen in Familial Mediterranean Fever (FMF). This familial type of amyloidosis, as one of the other types of familial amyloidosis, is genetically inherited and is found in specific population groups. In these two types of amyloidosis, deposits are found in several organs and are thus considered systemic amyloid diseases. Another type of systemic amyloidosis is found in long-term hemodialysis patients. In each of these cases, a different amyloidogenic protein is involved in amyloid deposition.
“Localized amyloidoses” are those that tend to involve a single organ system. Different amyloids are also characterized by the type of protein present in the deposit. For example, neurodegenerative diseases such as scrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob disease and the like are characterized by the appearance and accumulation of a protease-resistant form of a prion protein (referred to as AScr or PrP-27) in the central nervous system. Similarly, Alzheimer's disease, another neurodegenerative disorder, is characterized by neuritic plaques and neurofibrillary tangles. In this case, the plaque and blood vessel amyloid is formed by the deposition of fibrillar Aβ amyloid protein. Other diseases such as adult-onset diabetes (Type II diabetes) are characterized by the localized accumulation of amyloid in the pancreas. Amyloid deposits are present in pancreatic islets of up to 96% of patients with Non-Insulin Dependent Diabetes (NIDDM) at post-mortem. These fibrillar accumulations result from the aggregation of the islet amyloid polypeptide (IAPP), also known as amylin.
Once these amyloids have formed, there is no known, widely accepted therapy or treatment which significantly dissolves the deposits in situ.
Each amyloidogenic protein has the ability to organize into β-sheets and to form insoluble fibrils that get deposited extracellularly or intracellularly. Each amyloidogenic protein, although different in amino acid sequence, has the same property of forming fibrils and binding to other elements such as proteoglycan, amyloid P and complement component. Moreover, each amyloidogenic protein has amino acid sequences which, although different, will show similarities such as regions with the ability to bind to the glycosaminoglycan (GAG) portion of proteoglycan (referred to as the GAG binding site) as well as other regions which will promote β-sheet formation.
This suggests that amyloid fibrils are formed by a similar protein misfolding pathway and therefore therapeutic interventions to control their folding may be beneficial for all amyloid proteins. The associated proteins are the amyloid-β (Aβ) protein in AD and the islet amyloid polypeptide (IAPP) in Type-II diabetes. In both AD and Type-II diabetes, amyloid plays a key role which suggests that prevention of plaque formation will have significant therapeutic benefits.
In specific cases, amyloidotic fibrils, once deposited, can become toxic to the surrounding cells. As per example, the Aβ fibrils organized as senile plaques have been shown to be associated with dead neuronal cells and microgliosis in patients with Alzheimer's disease. When tested in vitro, Aβ peptide was shown to be capable of triggering an activation process of microglia (brain macrophages), which would explain the presence of microgliosis and brain inflammation found in the brain of patients with Alzheimer's disease.
In another type of amyloidosis seen in patients with Type II diabetes, and in patients with Type I diabetes post-transplantation, the amyloidogenic protein IAPP has been shown to induce β-islet cell toxicity in vitro. Hence, appearance of IAPP fibrils in the pancreas of Type II or Type I diabetic patients could contribute to the loss of the β islet cells (Langerhans) and organ dysfunction.
People suffering from Alzheimer's disease develop a progressive dementia in adulthood, accompanied by three main structural changes in the brain: diffuse loss of neurons in multiple parts of the brain; accumulation of intracellular protein deposits termed neurofibrillary tangles; and accumulation of extracellular protein deposits termed amyloid or senile plaques, surrounded by misshapen nerve terminals (dystrophic neurites). A main constituent of these amyloid plaques is the amyloid-β peptide (Aβ), a 40-42 amino-acid protein that is produced through cleavage of the β-amyloid precursor protein (APP). Although symptomatic treatments exist for Alzheimer's disease, this disease cannot be prevented nor cured at this time.
Amyloid-β and Alzheimer's Disease
Deposition of amyloid-β protein (Aβ) fibrils is an invariant feature of AD and is considered to be a major contributing factor to neuronal death and clinical dementia. Aβ is a proteolytic fragment of the β-amyloid precursor protein (βAPP), which accumulates in the neurophils as senile plaques and within cerebral blood vessels. These abnormal filamentous deposits co-localize with dystrophic neurites and reactive gliosis. Similarly, the cerebrovascular amyloid disruption of integral blood brain barriers may be a key factor in eliciting detrimental inflammatory responses leading to neuronal dysfunction. Aβ processing and deposition appear to be pivotal processes in AD pathogenesis which is supported by a number of lines of evidence. The strongest link is provided by the familial AD cases that result from mutations in either the APP or presenilin genes which display abnormal APP processing and herald an early onset and acceleration of the disease process.
Sequencing of Aβ a decade ago provided a catalyst for Alzheimer's research and quickly led to the cloning of the encoding gene. Aβ represents an internal sequence of the membrane-associated amyloid-β precursor protein (APP) that is organized into a large extracellular domain, a single transmembrane helix and a short cytoplasmic tail. Aβ constitutes 28 residues N-terminal to the extracellular-transmembrane interface as well as 12–14 residues of the transmembrane domain.
Previous investigations have demonstrated that synthetic Aβ and its various fragments will spontaneously assemble into amyloid-like fibrils.
In vitro peptide studies have identified important domains within the Aβ sequence, which facilitate aggregation and fibrillogenesis. For example, histidine residues at positions 13 and 14 appear to play a critical role in fibril formation as shown by the pH dependence of the β-conformation (Fraser, P. E. et al., J Mol Biol 244(1):64–73, 1994; and Wood et al., J Biol Chem 271:4086–4092, 1996). In addition, truncated and mutated peptides indicate that Aβ has two principal β-sheet domains spanning residues 10–25 and 30–42 possibly linked by α-turns. Interactions between both domains are essential for promoting aggregation as exemplified by the observation that small peptides derived from the 10–25 sequence (KLVFFA (SEQ ID NO. 25); residues 16–21) can inhibit fibril formation. Electron microscopy studies of amyloid plaques from AD tissue have revealed that fibrillar Aβ has an additional level of organization, which has been termed “protofilaments” (Serpell et al. J Mol Biol 300:1033–1039, 2000). These small, fibril substructures laterally aggregate to produce the final amyloid fibril.
The C-terminal sequence has been extensively investigated and significant differences between the Aβ1–40 and Aβ1–42 isoforms have been observed (Jarret et al., Biochemistry 32:4694–4697, 1993; and Jarret and Lansbury, Cell 73:1055–1056, 1993). Peptides containing the final two residues (Leu41-Val42) exhibit increased aggregation and a greater propensity towards fibril formation. The importance of the Aβ42 isoform is further demonstrated by its association with familial forms of AD. Mutations in either the APP or presenilin genes, which are related to early onset AD, all result in significantly elevated levels of the Aβ42, which may represent the nucleating “seed” that initiates Aβ polymerization.
Islet Amyloid Polypeptide and Diabetes
Amyloid deposits are present in pancreatic islets of up to 96% of patients with Non-Insulin Dependent Diabetes (NIDDM) at post-mortem. These fibrillar accumulations result from the aggregation of the islet amyloid polypeptide (IAPP) or amylin, which is a 37 amino acid peptide, derived from a larger precursor peptide, pro-IAPP. IAPP co-localizes and is co-secreted with insulin in response to β-cell secretagogues. This pathological feature is not associated with insulin-dependent diabetes and is a unifying characteristic for the heterogeneous clinical phenotypes diagnosed as NIDDM. The causal factors for islet amyloidosis and its role in the disease process have yet to be determined. However, longitudinal studies in cats and immunocytochemical investigations in monkeys have shown that a progressive increase in islet amyloid is associated with a dramatic decrease in the population of insulin-secreting β-cells and increased severity of the disease. More recently, transgenic approaches have strengthened the relationship of IAPP plaque formation and β-cell dysfunction, which indicates that amyloid deposition is a principal factor in Type-II diabetes.
Islet hyalinosis (amyloid deposition) was first described over a century ago as the presence of fibrous protein aggregates in the pancreas of patients with severe hyperglycemia (Opie, EL., J Exp. Med. 5: 397–428, 1990). Today, islet amyloid, composed predominantly of islet amyloid polypeptide (IAPP), or amylin, is a characteristic histopathological marker in over 90% of all cases of Type-II diabetes.
The mature IAPP molecule is a 37 residue peptide synthesized in the pancreas, and is co-localized with insulin in β-cell dense core secretory granules. Since IAPP is co-secreted with insulin, it has been suggested that IAPP plays a role in regulating blood glucose by controlling insulin secretion. The presence of soluble IAPP in the plasma itself is normally not problematic. In patients with Type-II diabetes, however, the accumulation of pancreatic IAPP leads to a buildup of IAPP-amyloid as insoluble fibrous deposits which eventually replace the insulin-producing β cells of the islet resulting in β cell depletion and failure (Westermark, P., Grimelius, L., Acta Path. Microbiol Scand, sect. A. 81: 291–300, 1973; de Koning, E J P., et al., Diabetologia 36: 378–384, 1993; and Lorenzo, A., et al., Nature 368: 756–760, 1994).
The fact that IAPP fibrillar aggregates are present in the pancreases of patients with severe Type-II diabetes and β-cell failure is evident. However, determining whether fibrillar IAPP is toxic to β-cells as well as the conditions that lead to aggregation of this peptide are currently areas of great interest. It has been suggested that differing levels of glycosylation may lead to a pool of peptide that is more apt to be involved in aggregation. Other studies have suggested that in Type-II diabetes, incomplete enzymatic processing of IAPP from its precursor pro-IAPP by the prohormone convertase PC2 may provide a level of aggregatable peptide needed for the “seeding” of amyloid fibrils. Still other studies have examined the properties contained in the amino acid sequence of human IAPP that make it prone to aggregation as compared to rodent IAPP which does not form typical amyloid fibrils (Johnson, K H., et al., N. Engl. J. Med 321: 513–518, 1989; and Moriarty, D F., Raleigh, D P. Biochemistry 38: 1811–1818, 1999).
IAPP amyloid has many features in common with cerebral amyloid formed in Alzheimer's disease from the amyloid-β ( Aβ) peptide. Both amyloid diseases are progressive and age-related and associated with irreversible deterioration in cellular function. Neither pathological conditions require synthesis of a mutated form of the peptide and both component peptides are derived from a larger precursor and form morphologically similar amyloid fibrils.
IAPP contains three principal domains that contribute to fibril formation. These domains have been identified by looking at different peptide fragments and also through the effects of the proline mutations in the rodent sequence, which does not form amyloid fibrils. The initial N-terminal domain, disulfide bridge (residues 2 and 7) is not critical to amyloid fibril formation.
Diseases caused by the death or malfunctioning of a particular type or types of cells can be treated by transplanting into the patient healthy cells of the relevant type of cell. This approach has been used for Type I diabetic patients. Often these cells are cultured in vitro prior to transplantation to increase their numbers, to allow them to recover after the isolation procedure or to reduce their immunogenicity. However, in many instances the transplants are unsuccessful, due to the death of the transplanted cells. One reason for this poor success rate may be IAPP, which can form fibrils and become toxic to the cells in vitro. In addition, IAPP fibrils are likely to continue to grow after the cells are transplanted and cause death or dysfunction of the cells. This may occur even when the cells are from a healthy donor and when the patient receiving the transplant does not have a disease that is characterized by the presence of fibrils.